An effective HIV-1 vaccine will likely require induction of broadly neutralizing antibodies (bnAbs). Many bnAbs exhibit poly/autoreactivity, and all accumulate high amounts of somatic mutations. While both traits appear important for bnAb specificity, they are likely related causally (with excess mutation capable of either creating or removing poly/autoreactivity), suggesting most or all bnAbs originate from B-cells that undergo negative selection at one point during affinity maturation. Furthermore, negative B-cell selection can occur through a myriad of potential mechanisms including apoptotic depletion, functional inactivation, or several distinct Ab variable heavy chain/variable light chain (VH/VL) modifying-processes, providing a global explanation for the general inability of HIV-1 infected subjects or vaccinees to elicit rapid and durable bnAb responses. Finally, poly/autoreactivity and high somatic mutation levels are potentially associated with pathogenicity, raising concerns that eliciting bnAbs with such traits may have clinically adverse effects. Thus, a critical question is whether poly/autoreactivity can be decoupled from broad neutralization specificity and whether the decoupling allows for a more efficient selection of bnAb B cell lineages during their maturation process. The overall objective of this proposal is to identify the minimal mutations required for poly/autoreactivity and neutralization breadth of nAbs and to assess whether poly/autoreactivity and neutralization breadth can be independently induced by immunization. The CH103 bnAb lineage has several features making it a unique, physiologically relevant model to explore this issue, including: a) its unmutated common ancestor (UCA) lacking poly/autoreactivity but developing these in later intermediate and mature Abs along with broad neutralization, b), experimental elucidation of its entire co-evolved viral and Ab clones, and c) relative to other CD4bs bnAbs, having less somatic mutations and being easier to elicit. We hypothesize that neutralization breadth and poly/autoreactivity are governed by different somatic mutations, and thus neutralization breadth can be decoupled from poly/autoreactivity for preferentially triggering desired neutralization clonal pathways. To test this central hypothesis, we will perform in vitro mutagenesis to systematically identify minimal mutations in CH103 lineage members responsible for broad neutralization and/or poly/autoreactivity. We will also use a novel CH103 UCA knock-in mouse model to determine in vivo evolutionary pathways of the CH103 bnAb lineage toward increased neutralization breadth and/or poly/autoreactivity and to evaluate the capacity of autologous Envs that can engage the CH103 bnAb lineage UCA and drive nAb maturation by gaining neutralization breadth and acceptable poly/autoreactivity. This study will lead to a better understanding of maturation pathways of the CH103 bnAb lineage, and results from this proposal will be critical in developing novel vaccine approaches for selectively eliciting bnAbs with desired specificities.